The use of fluorescent light in the microbiological diagnosis of chronic wounds, using the example of pressure ulcers

Introduction

Pressure ulcers (PUs) are characterised by local tissue injury to the skin over a bony prominence [1]. Their incidence is rapidly increasing and varies according to the world region. It has been estimated that PUs affect 12.8% (5.4/10,000 patient days) of people in Europe (10.8%) [2]. Búřilova et al. showed that in the Czech Republic, an average of 26,444 patients are diagnosed with PUs each year [3]. Treatment of PUs is a significant economic burden for both patients and the healthcare system. They increase mortality, worsen quality of life, prolong hospital stay, and change overall health outcomes [4, 5]. It has been shown that the mortality rate of patients with PUs is 2–6 times higher than in other complicated diseases [6, 7]. Dolanová et al. showed that in 2019, 30,590 people with PUs were recorded in the Czech Republic, of whom 40.9% died in the same year [8]. The most important predictors of the development of PUs are chronic progressive diseases, most often from cardiovascular diseases, the diagnosis of diabetes mellitus, and neurodegenerative diseases (dementia and stroke) [9]. The most significant factors contributing to the development of PUs are malnutrition associated with a low body mass index, high inflammatory markers (CRP, ESR), haemodynamic instability (low cardiac output, hypotension), and chronic complications significantly contributing to the development of inevitable PUs in immobile patients [10–12]. The healing of PUs can take several (sometimes a dozen or so) days, and in advanced clinical stages in an out-of-hospital setting it can take many months, and in some people complete healing may never occur [13].
Examination of the visual characteristics of wounds using modern devices allows for an accurate assessment of the condition of the wound (size, depth, type of tissue, blood flow, temperature, inflammation, and infection) and the surrounding skin [14]. Importantly, they can be helpful in the development of better and more effective methods of monitoring and treating PUs [15].
Very often, the surface of the PUs is significantly contaminated, and the bacterial load is usually high. Several tools have been developed to assess the clinical status of PUs (wound size, depth, condition of granulation tissue, and infection), such as DESIGN, the pressure sore status tool, the pressure ulcer scale for healing, and the Sussman wound healing tool [16]. There are also schemes available to detect the “classic signs” of bacterial infection, such as pain, failure to heal, purulent discharge, effusion, erythema, local temperature, and swelling [17–19]. However, the presence of classic signs of infection does not always clearly confirm wound infection. In several studies [20–22], 4 signs of infection (purulent discharge, inflammation, hypergranulation, and erythema) were shown to be ineffective in predicting bacterial loads > 10⁴ CFU/g. Le et al. showed that poor discriminatory power for signs of infection would have resulted in 84.7% (243/287) of patients with bacterial loads > 10⁴ CFU/g receiving inappropriate biocidal treatment [20]. On the other hand, pressure injuries are in a state of chronic inflammation, and these indicators can also occur in the absence of infection [23, 24]. During local wound assessment, tissue types associated with different stages of the wound healing process were identified. The wound healing processes (e.g. necrotic tissue, scab, granulation tissue) are visible to the naked eye. Their presence provides the clinician with valuable information about the current condition of the wound and its healing potential [25], which in turn guides the choice of treatment. In contrast, bacteria in the wound are not visible. The moist, nutrient-rich environment of PUs provides ideal conditions for the growth of microorganisms. Bacteria that inhabit their surface, under appropriate conditions, trigger a cascade of subsequent events that end in infection. The following are most frequently responsible for the development of infection: Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and Escherichia coli, and due to the large number of factors determining virulence, and production of toxins and enzymes, they are among the most dangerous pathogens infecting chronic wounds [26, 27].
Microbiological diagnosis of PUs using laboratory techniques is not ideal. Quantitative analysis performed on tissue biopsy is considered the gold standard for detecting high microbial burden in chronic wounds. However, it is invasive and often painful. Additionally, microbiological analysis is more time-consuming and expensive than semi-quantitative swab analysis, which is why culture remains a routine part of standard care [28]. However, the reliability of the test result depends on accurate wound sampling, and the waiting time is several days; as a result, many practitioners may decide not to collect samples from chronic wounds at all [28, 29]. Additionally, in the case of PUs, superficial swab cultures usually reflect bacterial colonisation rather than overt infection [30]. Therefore, at the point of care, the presence of bacteria at high burden is still inferred from clinical symptoms [17, 30, 31]. However, it is worth remembering that they do not provide information on the location(s) of high bacterial burden within and around the wound. In the meantime, the presence of a significant bacterial burden usually requires a bacteria-specific treatment plan, such as debridement or antimicrobial therapy [32, 33]. To overcome stagnant wound healing trends, improved methods for identifying and treating bacterial burden should be prioritised [20].
In recent years, attention has been paid to the use of fluorescent light (fluorescence imaging – FI) for diagnostic and therapeutic purposes [34, 35]. The MolecuLight i:X (MolecuLight, Canada) is a portable, noninvasive, and easy-to-use point-of-care FI device. The device enables rapid diagnostics to determine both the type and location of pathogens residing in the wound and on the skin in real time [36]. It detects bacteria, both planktonic and in the form of biofilm, but does not distinguish between them.
The signals produced, or colours, are tissue specific [37]; endogenous tissue components such as collagen fluoresce green, whereas clinically important bacteria producing metabolic byproducts such as porphyrins and pyoverdine fluoresce red and cyan (blue-green), respectively [38, 39]. Endogenous red fluorescent porphyrins emitted by bacteria have also been shown to allow visualisation and localisation of bacteria present at ≥ 10⁴ CFU/g and can be assessed on and below the wound surface, to a depth of ~1.5 mm [40]. It should be noted that many species of porphyrin-producing bacteria can colonise chronic wounds and produce red fluorescence, but Staphylococcus aureus is the most common bacterial species [41, 42]. Pyoverdine is unique to Pseudomonas aeruginosa; it is therefore the only bacterium that fluoresces cyan [41, 43]. The fluorescence image also provides a spatial pattern of bacterial burden, which creates a map of the bacteria in the wound and can be used by the clinician for targeted sampling, cleaning, bacterial removal, and other wound care methods [40]. Rapid microbiological diagnostics enable the implementation of appropriate local measures, such as wound bed cleansing and application of antimicrobial agents and dressings, even before the microbiological laboratory result is available [34, 44, 45].
The aim of our study was microbiological diagnostics of PUs to determine the species and location of pathogenic bacteria. Based on the results obtained from fluorescence imaging, targeted local measures (antibacterial) and effective debridement were implemented. This study also aimed to compare the results obtained from FI (MolecuLight i:X) with the results of bacterial wound cultures obtained in the microbiology laboratory.

Material and methods

We present two cases of patients with PUs in which we have used MolecuLight i:X images for more accurate microbiological diagnostics.
The description and the stages of the examination performed with the use of the MolecuLight i:X device
This study presents 2 cases of patients with PUs. In both cases, these were consultation visits to the Outpatient Department for Chronic Wound Management. After performing diagnostics and confirming the aetiology of the wound, clinical signs and symptoms of wound infection were assessed. The assessment was carried out by an interdisciplinary team of experts in wound treatment. Upon removal of the dressing from the wound, the topical treatment involved cleansing the wound bed and surrounding skin with the use of lavaseptic. Then a swab stick was moistened with natrium chloratum 0.9% (saline solution) and used to take a sample for microbiological testing. Levine swab samples were taken from the centre of the wound bed and sent for semi-quantitative culture analysis [46, 47]. Subsequently, a standard image of the wound was taken, placing the device at 8–12 cm from the wound. Standard images of the wound were captured in a conventional light setting, then the room was darkened and the fluorescence illumination mode was activated (violet light-emitting diodes – illuminating field of vision). The device used a range finder to make sure the images were captured within the optimal range (8–12 cm). The light sensor in the device indicated when the room was dark enough to capture fluorescence images. If switching off the light in the room did not result in sufficient darkness, the windows were covered with blinds. All the standard and fluorescent images were taken with the use of the device (MolecuLight i:X). The device in question consists of camera sensor, fluorescence optical emission filter, and 2 light emitting diodes, which emit a narrow band of 405-nm violet-coloured excitation light. Red or cyan fluorescence signals indicate the presence of bacteria at loads > 10⁴ CFU/g (moderate-to-heavy growth) [20, 41, 48]. Red fluorescence is emitted by porphyrins, endogenous fluorophores produced by bacterial species such as Staphylococcus aureus [34]. Cyan fluorescence signal is attributed to pyoverdines, which are uniquely produced by Pseudomonas aeruginosa [49, 50]. These signals are produced by both planktonic as well as biofilm-encased bacteria [28, 34, 51]. The fluorescence signals were displayed on a digital touch screen and immediately interpreted by the clinician. The procedure with the use of the MolecuLight i:X device was developed based on current literature. Next, depending on the result obtained from the fluorescence image, appropriate treatment methods were used, such as debridement of the wound surface, disinfection, optimal dressing choice, and/or causal treatment in the form of short-stretch compression bandages. These methods were in accordance with current recommendations for venous ulcer care [52].

Case reports

Case 1

A 62-year-old man was admitted to the Chronic Wound Clinic for a consultation due to the lack of progress in the treatment of a stage 3 PUs. Two years previously, the patient had surgery due to intestinal obstruction, and he developed a PUs during his hospital stay. The man had a temporary colostomy, and the presence of the PUs prevented surgery to restore the continuity of the large intestine.
Description of the PUs – the pressure ulcers was located in the sacrum area. The wound surface was 22 cm², depth – 1 cm, and the wound edges were rolled up, separated from the wound, with traces of maceration. The wound bed consisted mainly of pale granulation tissue. The pressure ulcer did not show typical signs of wound infection. Figure 1 A shows the image in white light recorded by a wound imaging device. Figure 1 B shows areas of red fluorescence suggesting the presence of surface or subsurface bacteria located on the wound edges. The wound surface showed the presence of Streptococcus oralis < 10³ CFU/g, a physiological skin microbiota. The image obtained using fluorescent light confirmed the microbiological result. Microbiological diagnostics of subsurface bacteria is difficult, and thanks to fluorescent diagnostics we learned the cause of the healing arrest and were able to implement effective therapeutic measures (systematic mechanical cleansing of the wound edges and antimicrobial agents). Due to the long distance from his home to the Chronic Wound Treatment Clinic, the patient continued treatment at home. After 4 months, he informed us by phone that the PUs had healed, and he underwent a procedure to restore the continuity of the digestive tract.

Case 2

A 76-year-old woman was admitted to the Neurology Clinic due to a cerebral infarction. The patient was lying on the floor at home for several hours after the neurological incident. Description of the PUs – A pressure ulcer developed in the area of the left hip joint as a result of contusion and/or tissue compression (Fig. 2 A). The wound surface was 65 cm2, depth approximately 4 cm, and the wound bed was covered by 70% necrosis. Microbiological analysis of a tissue sample directed to the area of red fluorescence showed the presence of Morganella morganii > 10⁴ CFU/g, Serratia marcescens > 10³ CFU/g, Klebsiella pneumonia < 10⁴ CFU/g. Based on information from FI (Fig. 2 B) and the general clinical picture of the patient, targeted antibiotic therapy and local biocidal activities were initiated. Modern imaging technology indicated areas requiring monitoring and cleaning (bacteria and biofilm), thanks to which it was possible to assess the effectiveness of the actions taken. Pictures were taken during the first visit (Figs. 2 A, B), and then after 7 days (Figs. 2 C, D) of systematic cleansing of the wound surface with agents intended for infected wounds.

Discussion

Early identification and appropriate treatment with FI can prevent many complications, such as acute cellulitis, abscess, hospitalisation, and/or surgical intervention, thereby reducing healthcare costs [53, 54]. In the future, FI of chronic wounds may initiate a paradigm shift among wound care providers. The use of a fluorescent imaging device has several advantages in both outpatient and inpatient care [28]. It is simple to use, requires little training, and can be used by all healthcare professionals. When combined with best clinical practice, the device can assist in decision-making regarding general and local treatment, such as the selection of biocidal agents and specialised dressings [55]. It has been proven to be rapid, with the procedure taking no more than a minute per patient, which is less than conventional clinical assessment. As demonstrated by Blackshaw et al., results are shown in real time, and treatment decisions are made at the patient’s bedside [56]. Wu et al. provide a detailed description of the use of auto-FI imaging as an aid during wound debridement to detect potentially pathogenic bacteria, changing the treatment decision-making process [37]. Our case reports illustrate different aspects of the use of the device for FI and more accurate diagnostics.
In case 1, we presented a patient with a stage 3 PUs. The patient came to our centre because the wound had not healed for 2 years. Lee E. showed that in Korea (n = 184), the probability of category/stage I or II Pus healing in any given month was 5.12%, and only 10% of PUs healed completely within 12 months [57]. A second cohort study conducted in the United Kingdom showed complete healing within 12 months in 69% of category/stage II PUs, 41% of category/stage III PUs, and 21% of category/stage IV PUs, with a mean healing time of 5.4 months [58]. It seems that objective and precise diagnosis and monitoring of PUs healing may improve the healing conditions and thus shorten the duration of treatment. In our case, the PUs showed no clinical signs of infection. In our study we used the MolecuLight i:X device to guide microbiological sampling sites, and based on the results, we recommended effective local interventions. Although the PUs showed no clinical signs of infection, FI revealed the presence of subsurface bacteria. With FI, we were able to identify the cause of delayed healing of the PUs. Similar results were shown in a study conducted in the United States in 14 outpatient advanced wound care centres. Wounds were assessed for clinical signs of infection and then photographed using fluorescence. Biopsies were taken to confirm bacterial burden. A total of 350 patients completed the study (138 diabetic foot ulcers, 106 venous leg ulcers, 60 surgical sites, 22 PUs, and 24 other). Approximately 287/350 wounds (82%) were found to have bacterial burdens > 104 CFU/g, whereas 85% of these wounds were misdiagnosed based on clinical assessment of infection. FL significantly increased bacterial detection (> 10⁴ CFU/g), which was consistent across wound types (p < 0.001). FL information modified treatment plans (69% of wounds), influenced wound bed preparation (85%), and improved overall patient care (90%), as reported by the study clinicians [20]. Moelleken et al. [59] showed that after a single mechanical debridement, red fluorescence in the wound bed was reduced by 99.4%, from 10.44% of the total wound area to 0.06% (p < 0.001). In this case study, we demonstrated the efficacy of fluorescent light in detecting subsurface bacteria that are not detected by traditional swabs. Although no pathogens were cultured, FI identified areas of increased bacterial burden below the wound edge. The efficacy of this imaging device has been previously demonstrated in other studies [37, 56].
In the second case, we presented a patient with a stage 3 PUs. The patient was consulted by our team to determine the direction of PUs treatment. We performed an imaging study using FI to detect places with the highest bacterial load, and we took samples from them for microbiological examination. We also indicated places on the wound surface that should be paid special attention during debridement. We used the MolecuLight i:X device during the second consultation to assess the effectiveness of the actions taken, both general and local. We showed a significant reduction in the areas covered in red, i.e. a reduction in bacterial load. Raizman et al. conducted a similar observation, but with the participation of 22 patients with diabetic foot ulcers, and assessed, among others, the effectiveness of debridement using FI. They showed that bacterial fluorescence decreased in all cases after additional, targeted debridement, but it could not always be completely removed by debridement alone. In these cases, additional antimicrobial strategies were implemented. Of the 20 wounds evaluated, 3 (15%) were not further debrided because in some cases, more aggressive debridement could have caused, among other things, bleeding [60]. Another study found that the average weekly change in wound area was a 6% increase when bacterial fluorescence was present and changed to a 27% decrease in the wound area when bacterial fluorescence was eliminated through targeted debridement and other antimicrobial strategies. The fluorescence information in this study provided evidence-based documentation to establish the appropriate level of debridement [61]. Validation is important to ensure appropriate clinical implementation. In this study, the accuracy, and inter- and intra-user variability of the digital wound imaging device software was assessed using laboratory models and clinical images. The ease of wound measurement and implementation of FI in the clinical setting was then assessed in a clinical study of 50 wounds. This study further documented the high prevalence of bacterial fluorescence in the general wound population, which is significantly underestimated by standard assessment of clinical signs and symptoms of care [22, 30, 48]. Finally, bacterial FI has been used to assess bioburden before and after debridement, and to inform and document when additional and more targeted debridement is needed to remove bacterially burdened tissue. This compilation of work demonstrates that a bacterial FI device can be easily and reproducibly implemented for real-time wound assessment at the point of care to improve wound documentation and guide treatment in the clinical setting.
Similarly, in our observation we showed that wound cleansing reduced the number of bacteria in the wound but did not eliminate them completely.

Limitations

Interpretation of the results of this single-centre study must be considered in the context of the study limitations. Due to the pilot, exploratory nature of this study, the sample size was small, and the study was not statistically significant.

Conclusions

The significant bacterial burden of PUs is often underestimated. Incorporating the noninvasive diagnostic procedure of fluorescence imaging into wound assessment could significantly improve detection of pathogens and provide information on bacterial localisation at the point of care. This represents a paradigm shift in wound assessment in which wound care providers have immediate information on microbiological status to guide treatment selection and determine the frequency of reassessment to assess the effectiveness of selected point-of-care treatments. We presented only 2 cases in our paper; we will continue with our study because the results from international scientific sources are promising.

Disclosures

1. Institutional review board statement: Not applicable.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References